Post-transcriptional regulation of the heat stress response in plants – HEAT-ADAPT

During this project, we will produce biological material and generate data that will help to better understand the biological role of mRNA degradation induced by heat stress and the phenomenon of heat tolerance in plants. A minimum 40 single or double mutant lines will be constructed during the project. All the mutant lines will be phenotyped in at least five different thermal stress regimes. A thermotolerance deficit phenotype will be searched in at least three different development stages (seedlings to test the survival and growth defects, seeds to test germination defects and flowers to test fertility defects). This genetic approach combined with phenotypic screening will provide a comprehensive directory of actors of mRNA degradation that are requested to induce heat-stress mediated decay. As the proteins involved in mRNA degradation processes are highly conserved in all plants, each actor identified by this approach represents a potential target for future breeding programs to increase the thermotolerance properties of crop plants. We will also identify mRNA populations that are targeted by this degradation mechanism. This information, coupled with our epigenomics approach will allow us to better understand the principles of mRNA targeting to degradation and thus pave the way for the engineering of heterologous mRNAs which could be degraded or not following a rise in temperature. This ability to target a specific recombinant mRNA degradation for degradation based only on temperature is unprecedented and has great potential for technological use that may be patented under this project.

1. Identification of a co-translational decay process induced during heat-stress linking mRNA stability to a protein quality control mechanism. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of ‘non-aberrant’ mRNAs.

2. Identification of a heat-specific post-translational modification of a reader of mRNA m6A methylation status, that could regulated the stability of mRNAs during heat stress.

3. Set up of an improved protocol to establish the plant mRNA methylome.

4. Identification of a key role for HSP101 in the recovery period. HSP101 is important to target stored mRNAs back to translation after heat stress. A paper describing this work is in preparation.

The project has already provided some very interesting results and the large-scale analyses coming up soon (methylome and half-life measures of mRNA) should allow key breakthrough in the field.

1. Heat-induced ribosome pausing triggers mRNA co-translational decay in Arabidopsis thaliana
Remy Merret, Vinay K. Nagarajan, Marie-Christine Carpentier, Sunhee Park, Jean-Jacques Favory, Julie Descombin, Claire Picart1, Yee-yung Charng, Pamela J. Green, Jean-Marc Deragon and Cécile Bousquet-Antonelli. Nucleic Acids Research, 2015, Vol. 43, No. 8 4121–4132. nar.oxfordjournals.org/content/43/8/4121.full.pdf+html.

2. Heat induces storage of ribosomal protein mRNAs to enchance ribosomes production during post-stress recovery in Arabidopsis
Rémy Merret, Marie-Christine Carpentier, Jean-Jacques Favory, Claire Picart, Cécile Bousquet-Antonelli, Pascal Tillard, Laurence Lejay, Jean-Marc Deragon, and Yee-yung Charng. In preparation.

The capacity of plants to survive exposure to a variety of environmental stresses is of prime importance for food production, especially in the context of global warming. Global climate change is expected to result in a 1.5 to 5.8°C increases in temperature by 2100 and crop yields are predicted to decrease approximately 10% for every one-degree increase in temperature (USDA Release no. 501.09, 2009). Exceptionally high summer temperature is already leading to strong reduction in crop yield and plant are more then ever exposed to extremes of temperature that, combined to other stresses such as drought, is negatively impacting agricultural production worldwide. How climatic variations impact plant life cycles is therefore an urgent question to address in order to improve stress tolerance and adapt agriculture to future rises in temperature. Currently, we have a limited knowledge of the basic molecular mechanisms by which plants survive to stress. Therefore, studying the regulation of gene expression in response to environmental cues is fundamental to understand how plants grow and develop. Recent studies indicate that post-transcriptional regulation of gene expression plays a vital role in stress response and that a radical reprogramming of mRNA decay could be involved. We recently discovered that plant drastically reprogram mRNA decay during heat stress. Indeed, we observed that the RNA-binding protein LARP1 associates during heat stress (15 min at 38°C) with the 5’-3’ exonuclease XRN4 to set up a massive heat-induced mRNA decay process that apparently targets more that 4500 mRNAs in Arabidopsis seedlings. LARP1 is specially required to address XRN4 to polysomes during heat stress, suggesting that part of the degradation is directly initiated on mRNAs engaged in translation. We have also shown by preliminary work that xrn4 mutant plants are clearly affected in their capacity to survive to at least one heat stress regime. Overall, these results strongly suggest that plants regulate the heat stress response at the post-transcriptional level by inducing an essential targeted mRNA decay process. Following this pioneer work, many questions still remain on the biological properties of this Heat-Stress Mediated Decay (H-SMD) including: 1) what is the complete repertoire of Arabidopsis thaliana mRNAs targeted by H-SMD? 2) what other molecular actors are involved ? 3) How mRNAs are selected to be targets of H-SMD and, 4) what is the physiological importance for the plant of this process. In this program we will address these questions by combining biochemical, genetic and phenotypic approaches but also by using new genome-wide technologies. Indeed, using an original pulse-chase non-radioactive labeling method, we will perform a genome-wide analysis of plant mRNAs half-life at 20°C and 38°C. We will also evaluate the general importance of RNA epigenetic modifications (i.e. the presence of patterns of N6-methyladenosines) for mRNA stability at 20°C and 38°C. Most studies so far addressed the plant thermotolerance response at the transcriptional levels. A major originality of this work is to postulate that a post-transcriptional process is also determinant for the plant to acquire thermotolerance properties. Our four interrelated workpackages will allow determining the physiological importance of mRNA decay for plant survival to heat stress. The two partners of the project are highly complementary and have already collaborated on the initial characterization of the heat-induced decay pathway. Partner 1 has a strong expertise in molecular studies of mRNA metabolism and epigenetic regulations. Partner 2 has strong expertise in phenotyping methods to explore thermotolerance diversity and is an international expert of the plant heat stress response. Bringing together these two partners will lead to a tighter integration of the research efforts in this exciting and innovative area of RNA epigenomics and plant stress adaptation.